The enzymes Hydroxymethyl Coenzyme A Reductase (HMGCoAR) and Acyl CoA (cholesterol) Acyltransferase (ACAT) control two metabolically interrelated processes of cholesterol synthesis and esterification, respectively. The activities of both enzymes are rapidly affected by reversible phosphorylation (HMGCoAR is inhibited, ACAT is stimulated) and they are also subject to long-term effects on rates of synthesis. Since there is presently controversy in literature as to the role of phosphorylation of the enzyme in the metabolic regulation of sterol synthesis and lack of information on this process in ACAT, we have assayed in the same experiments cholesterol synthesis and HMGCoAR activity in human skin fibroblats. ACAT was studied in fibroblasts and rat liver microsomes. It was shown that at low concentrations and within a period not exceeding 30 min 7-ketocholesterol and 25-OH-cholesterol caused in parallel and to a similar degree inhibition of incorporation of labeled acetate into nonsaponifiable lipids and reversible phosphorylation of HMGCoAr without diminishing concentration of the enzyme as measured by its total activity. ACAT activity was greatly depressed following incubation with phosphoprotein phosphatase. Intubation of rats with mevalonolactone rapidly stimulated microsomal ACAT.